Comparative effects of amphetamine-like psychostimulants on rat hippocampal cell genesis at different developmental ages

NeuroToxicology 56 (2016) 29–39

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NeuroToxicology

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Comparative effects of amphetamine-like psychostimulants on rat hippocampal cell genesis at different developmental ages Rubén García-Cabrerizo, M. Julia García-Fuster* Neurobiology of Drug Abuse Group, IUNICS/IdISPa, University of the Balearic Islands, Palma de Mallorca, Spain

A R T I C L E I N F O

Article history: Received 1 June 2016 Received in revised form 28 June 2016 Accepted 28 June 2016 Available online 29 June 2016 Keywords: Adolescence MDMA Methamphetamine D-Amphetamine Hippocampus Neurogenesis

A B S T R A C T

The aim of this study was to compare the effects of amphetamine-like psychostimulant drugs (i.e., MDMA, methamphetamine, D-amphetamine) on rat hippocampal cell genesis at different developmental ages (i.e., early adolescence vs. young adulthood) to determine if there were periods of vulnerability to drug-induced brain changes. Although adolescence is a period of great vulnerability to the neurochemical effects of specific drugs of abuse, several reports suggest that adult rats are more susceptible than adolescents to the negative effects of these drugs. The main results suggest that the effects of these amphetamine drugs on cell genesis depend on the rat’s developmental age, with the young adult period being more sensitive than the early adolescent one. In particular, MDMA and methamphetamine, but not D-amphetamine impaired hippocampal cell genesis (i.e., cell proliferation and cell survival) in young adult rats. These effects were dependent on the accumulative dose administered, as they were only observed with the highest dose tested (12 pulses of 5 mg/kg over 4 days: 60 mg/kg total). The present results extend previous reports on adolescent insensitivity (i.e., better adaptation) to amphetaminedrugs and suggest for young adult rats certain degree of hippocampal damage that may mediate some of the addiction-like behaviors that depend on this brain region. Moreover, the present results, in line with previous data, suggest a possible role for the neuroplasticity marker BDNF and serotonin in regulating cell survival, as mBDNF protein regulation paralleled hippocampal cell survival and 5-HT2C-receptor content in young adult rats treated with these psychostimulant drugs. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction Amphetamine-like psychostimulants, including 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine and Damphetamine are widely abused drugs (UNODC, 2014) for their stimulant and euphoric effects. Typical recreational use is often characterized by a binge administration (i.e., repeated frequent administration during a short period of time). In contrast to MDMA, methamphetamine and D-amphetamine are also available under medical prescription and are used in the treatment of attention deficit hyperactivity disorder, narcolepsy and weight control (reviewed in Steinkellner et al., 2011). As these drugs are commonly abused by adolescents and young adults (SAMHSA, 2002) but are known to affect their brain function and behavior differently, the age of drug exposure will be crucial for the neurotoxic outcome (Vorhees et al., 2005; Teixeira-Gomes et al.,

* Corresponding author at: IUNICS, University of the Balearic Islands, Cra. de Valldemossa km 7.5, E-07122 Palma de Mallorca, Spain. E-mail address: [email protected] (M. J. García-Fuster). http://dx.doi.org/10.1016/j.neuro.2016.06.014 0161-813X/ã 2016 Elsevier B.V. All rights reserved.

2015). Although adolescence is a period of great vulnerability to the neurochemical effects of specific drugs of abuse (Spear, 2007), several reports suggest, in fact, that adult rats are more susceptible than adolescents to the negative effects of these drugs (Brunell and Spear, 2006; Teixeira-Gomes et al., 2015). Amphetamine-like psychostimulants interact in the central nervous system with monoamine transporter sites with different affinities: MDMA shows greater affinity for norepinephrine and serotonin transporter over dopamine transporter while methamphetamine and D-amphetamine have more potent actions on norepinephrine and dopamine release than serotonin (reviewed in Teixeira-Gomes et al., 2015). Previous work have ascertained numerous interacting mechanisms and neural consequences that might contribute to the known damage produced by these amphetamine-like psychostimulants (reviewed in Steinkellner et al., 2011; Teixeira-Gomes et al., 2015), such as monoaminergic terminal damage, excitotoxicity, oxidative stress and metabolic compromise, that in the long term could affect mental health and well-being. However, much work still needs to be done to fully elucidate the neurochemical changes induced by these drugs in

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precise brain regions, with the dosage regimen and the species under study being major variables (e.g., Green et al., 2003). In particular, the hippocampus is highly sensitive to drug abuse during the developmental period (e.g., Izenwasser, 2005) as it continues to undergo structural and functional changes throughout adolescence and into adulthood, which are vital for its maturation and neuronal function. Different drugs of abuse, including amphetamines, are known to induce hippocampal toxicity by impairing neurogenesis in adult rats (i.e., diminish cell genesis at different stages of the addictive cycle; reviewed in Canales, 2010; Mandyam and Koob, 2012). Interestingly, only a few studies have evaluated hippocampal cell genesis during adolescence and have proven critical windows of vulnerability to certain drugs such as cocaine (García-Cabrerizo et al., 2015) and alcohol (Crews et al., 2006) as compared to other age periods. Therefore, this study is a direct comparison of the effects of MDMA, methamphetamine and D-amphetamine on cell genesis (i.e., cell proliferation and cell survival) in the hippocampus following the same dosing regimen at different developmental ages (early adolescence vs. young adulthood) to determine if they were periods of vulnerability to drug-induced brain changes. Previous data supports a critical role of brain-derived neurotrophic factor (BDNF) and serotonin in the survival and preservation of neurons in the brain (Mattson et al., 2004; Foltran and Diaz, 2016). Thus, the possible regulation of this neuroplasticity marker and that of 5HT2C receptor, a serotonin receptor subtype present at higher densities in the hippocampus (Berumen et al., 2012) and with a known role in promoting neurogenesis (Klempin et al., 2010), will also be evaluated following the administration of these psychostimulant drugs. 2. Materials and methods 2.1. Rats Male Sprague-Dawley rats (n = 108; from Charles River, L’Ambresle, France) were purchased at weaning (post-natal day, PND 21, n = 54) or mid-late adolescence (PND 42, n = 54) (see Teixeira-Gomes et al., 2015 for rat characterization of developmental ages). Each

psychostimulant drug treatment (1 or 4 days of MDMA, methamphetamine, D-amphetamine) was performed at different points in time in separate experiments and therefore using different colonies of rats (n = 36 rats per drug: n = 18 per age) from the same vendor. Every drug-treated group was randomly allocated and paired with controls at any given developmental time of study. Rats were housed in standard cages in controlled environmental conditions (22
C, 70% humidity, and 12-h light/dark cycle, lights on at 8:00 a.m.) with ad libitum access to a standard diet and tap water. Prior to any experimental procedure, rats were habituated to the experimenter by being handled daily for two days. All animal care and experimental procedures were comply with the ARRIVE guidelines (Kilkenny et al., 2010) and were conducted according to standard ethical guidelines (European Communities Council Directive 86/ 609/EEC) and approved by the Local Bioethical Committee (UIBCAIB). All efforts were made to minimize the number of rats used and their suffering. 2.2. Drug treatment and tissue collection As depicted in Fig. 1a, groups of randomly allocated rats from two different developmental ages were pretreated for 3 consecutive days (PND 27–29 or PND 48–50) with 5-bromo-20 -deoxyuridine (BrdU, 2  50 mg/kg, i.p.; Calbiochem, USA) delivered at 12-h intervals as previously described (García-Fuster et al., 2010; García-Cabrerizo et al., 2015). Then, rats received a binge paradigm (5 mg/kg, every 2–3 h, 3 times per day, i.p.) during 1 (PND 36 or PND 57: 3 pulses, total dose of 15 mg/kg) or 4 consecutive days (PND 33–36 or PND 54–57: 12 pulses, total dose of 60 mg/kg; see Fig. 1a) with the amphetamine derivatives MDMA (kindly provided by ‘Agencia Española de Medicamentos y Productos Sanitarios, Ministerio de Sanidad, Política Social e Igualdad’, Spain), (+)-methamphetamine hydrochloride (Sigma-Aldrich, MO, USA) or D-amphetamine sulfate (Sigma-Aldrich, MO, USA). As shown in Fig. 1a rats exposed to the 1 day psychostimulant regimen received saline (0.9% NaCl, 1 ml/kg, every 2–3 h, 3 times per day, i.p.) for 3 days followed by one day of amphetamine drug treatment on PND 36 or PND 57. Notably, one rat from the 4 days MDMA treatment group died following the first day of treatment (PND 54).

Fig. 1. Experimental design. (a) Adolescent and young adult rats were treated at the indicated post-natal days (PND, n = 36 rats per drug: n = 18 per age: n = 6 per treatment group). Initially, rats were treated with BrdU (2 pulses  50 mg/kg, i.p., for 3 days). Then, control rats received saline (0.9% NaCl, 1 ml/kg, every 2–3 h, i.p., for 4 days) while other rats received amphetamine-like psychostimulants (3  5 mg/kg, every 2–3 h, i.p.) for 1 or 4 days. Rats were sacrificed 24 h after the last dose (PND 37 or PND 58). (b) Schematic representation of the molecular markers evaluated in the hippocampus by immunohistochemistry or western blot analysis.

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The comparative individual (5 mg/kg, 3 vs. 12 pulses) and accumulative doses (15 vs. 60 mg/kg) were selected on a mg/kg basis (e.g., Schaefer et al., 2008), so inferences about relative magnitude of effect among drugs could be made. Another possibility would have been to match the doses by initial hyperthermia, although this has proven difficult, as core body temperature did not remain comparable throughout the course of methamphetamine and D-amphetamine treatments (Vorhees et al., 2011). Additionally, doses were chosen based on previous studies in which these amphetamines derivatives were shown to induce changes in brain neurochemistry and/or neurotoxicity in Sprague-Dawley rats (see review, Green et al., 2003; TeixeiraGomes et al., 2015) and not taking into account the high accumulative dose selection in comparison to human exposure. In any case, based on pharmacokinetic/dynamic data and studies on dose translation from animal to humans (Reagan-Shaw et al., 2008), an approximate four-fold higher dose is required in rats to produce a similar peak blood plasma exposure to that seen in humans (Green et al., 2009), therefore justifying the doses of amphetamine-like drugs used in the present study (i.e, 5 mg/kg per injection) as compared to the typical dose consumed by humans (average of 1–2 mg/kg). Control rats received saline (0.9% NaCl, 1 ml/kg, every 2–3 h, 3 times per day, i.p.) during all treatment days. Rats were weighted the days of BrdU administration (PND 27– 29 or PND 48–50) and throughout drug treatment (PND 33–36 or PND 54–57). All rats (a total of 107) were killed at the appropriate times for each amphetamine-like drug (35–36 rats for each drug paired with controls) by decapitation without anesthesia 24 h after the last treatment dose (PND 37 or PND 58). All the extracted brains were cut sagitally to separate both hemispheres. The left half-brain was quickly frozen in 30
C isopentane and stored at 80
C until further processing. For each animal, 30 mm sections were cryostat cut and slide-mounted throughout the entire hippocampal extent (1.72 to 6.80 mm from Bregma) and kept at 80
C until cell genesis markers (i.e., Ki-67 for cell proliferation and BrdU for the survival of 8–10 days old cells; García-Fuster et al., 2010) were analyzed by immunohistochemistry (see Fig. 1b). The hippocampus was freshly dissected from the right half-brain, rapidly frozen in liquid nitrogen, and kept at 80
C until the neuroplasticity markers BDNF and 5-HT2C receptors (see Fig. 1b) were analyzed by western blot experiments. 2.3. Immunohistochemistry analysis Ki-67 was used to determine the rate of recent cell proliferation (all dividing cells within a cell cycle time of 25 h; Cameron and McKay, 2001) in hippocampal tissue sections (30 mm) that were post-fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, USA). Antigen retrieval (10% sodium citrate pH 6.0, 90
C, 1 h) was followed by blocking sections in peroxidase solution (0.3%, 30 min) and bovine serum albumin (BSA; Sigma-Aldrich, USA) containing 1% goat serum (Sigma-Aldrich, USA) and 0.05% Triton X-100 (Sigma-Aldrich, USA; see García-Fuster et al., 2010; GarcíaCabrerizo et al., 2015). Sections were then incubated overnight with polyclonal rabbit anti-Ki-67 (1:40000; kindly provided by Drs. Huda Akil and Stanley J. Watson, University of Michigan) followed by incubation in biotinylated anti-rabbit secondary antibody 1:1000 (Vector Laboratories, Burlingame, CA). Bound antibody was detected with Avidin/Biotin complex (Vectastain Elite ABC kit; Vectors Laboratories, USA) and the chromogen 3,30 diaminobenzidine (DAB; Sigma-Aldrich, USA). BrdU is a synthetic thymidine analog incorporated into newly synthesized DNA strands of actively proliferating cells (Wojtowicz and Kee, 2006). Therefore, the age of the BrdU labeled

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cells at the time of euthanasia determines the developmental stage of the newly born cell (i.e., proliferation, differentiation, maturation). In our experimental design, pretreatment of rats with BrdU allowed to study drug effects on the survival of 8– 10 days old cells. Tissue was post-fixed in 4% PFA for 1 h, rinsed with in-lab prepared phosphate buffered saline (PBS)and washed in 0.3% peroxide (García-Fuster et al., 2010). Sections were then placed in 50% formamide-2X saline sodium citrate (SSC) at 65
C for 2 h, rinsed in 2X SSC and exposed to 2 N HCl at 37
C during 30 min for DNA denaturation. Afterwards they were incubated for 10 min in 0.1 M boric acid at room temperature followed by rinsing in PBS, blocking with BSA and overnight incubation with polyclonal rabbit anti-BrdU (1:20 000; kindly provided by Drs. Huda Akil and Stanley J. Watson, University of Michigan). Finally, sections were incubated with anti-rabbit secondary antibody 1:1000 (Vector Laboratories), followed by Avidin/Biotin complex amplification and DAB reaction for visualization of signal. All sections were counterstained with cresyl violet (SigmaAldrich, USA), dehydrated through graded alcohols, immersed in xylene and coverslipped (Permount1 mounting medium; Fisher Scientific, USA). The number of immunostained positive cells was evaluated under blinded conditions in every 8th section throughout the extent of the hippocampus (1.72 to 6.80 mm from Bregma) with a Leica DMR light microscope using a 63 objective lens and focusing through the thickness of the section (30 mm). Table 1 summarizes the number of tissue sections as well as the area of the dentate gyrus (mm2) analyzed for each cell marker (Ki67, BrdU), at each developmental age (PND 37, PND 58) and treatment group. The number of proliferating (Ki–67+) or surviving (BrdU+) cells in the dentate gyrus was expressed in relation to the overall quantified area (mm2), which was measured with a densitometer (GS-800 Imaging Calibrated Densitometer, BioRad). This quantification method allows the comparison among groups of treatment with different areas of analysis (García-Cabrerizo et al., 2015), which is particularly relevant when comparing different developmental ages (PND 37 and PND 58) or different cell markers (Ki-67 and BrdU labeling). 2.4. Western blot analysis Total brain proteins (40 mg) from rat hippocampus homogenates were resolved by electrophoresis on 10–12% SDS–PAGE minigels (Bio-Rad Laboratories, Hercules, CA, USA) and then transferred to nitrocellulose membranes that were incubated overnight at 4
C in blocking solution containing the appropriate primary antibody (García-Cabrerizo and García-Fuster, 2015) whose vendors and dilution conditions were the following: (1) Santa Cruz Biotechnology (CA, USA): anti-BDNF (N-20) (1:2500), anti-5-HT2C (D-12) (1:500); and (2) Sigma-Aldrich (MO, USA): anti-ß-actin (clone AC-15) (1:10 000), anti-a-tubuline (clone B-51-2) (1:2000). The secondary antibody (anti-rabbit or antimouse IgG linked to horseradish peroxidase) was incubated for 1 h at room temperature (1:5000 dilution; Cell Signaling). Immunoreactivity of target proteins was detected with ECL reagents (Amersham, Buckinghamshire, UK) and signal of bound antibody was visualized by exposure to autoradiographic film (Amersham ECL Hyperfilm) for 1–60 min, which was quantified by densitometric scanning (GS-800 Imaging Calibrated Densitometer, Bio-Rad). The amount of target proteins for each psychostimulant treatment group (MDMA, methamphetamine or D-amphetamine) was compared in the same gel with that of its respective control rats for each developmental age (PND 37 or PND 58). The quantification procedure was assessed 3–6 times in different gels (each gel with different brain samples from control and psychotimulant-treated rats). Percent changes in

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Table 1 Number of sections analyzed and area (mm2) quantified in the dentate gyrus region of the hippocampus for each treatment group. Data are means  SEM of n rats per group.

immunoreactivity with respect to control samples (100%) at each age time-point (PND 37 or PND 58) were calculated for each treated rat in various gels, and the mean value was used as a final estimate. As the content of ß-actin was slightly decreased by chronic MDMA in the hippocampus at PND 37 (11  2%, p < 0.05), a-tubulin was evaluated and quantified as a loading control as its content was not altered by any treatment conditions. 2.5. Data and statistical analysis Data were analyzed with GraphPad Prism, Version 6. Results are expressed as mean values  standard error of the mean (SEM). Each psychostimulant drug (MDMA, methamphetamine, Damphetamine) and each developmental age (PND 37 and PND 58) were evaluated separately, as different colonies of rats were used at different points in time for each experiment. The effects of each drug on body weight were evaluated using two-way

repeated measures ANOVAs, in which Treatment (control vs. drug) and Day of Treatment (PND 27–36 or PND 48–57) were treated as independent variables, followed by Bonferroni’s multiple comparison tests when appropriate. For the immunohistochemistry and western blot data analysis, two-tail Student’s t-tests were used to ascertain statistical differences between groups: PND 37 vs. PND 58 when comparing basal cell genesis at different developmental ages; and control vs. drug treatment for ascertaining percent differences between each drug treatment and its respective PND control group. Finally, as the main effects on cell genesis markers were observed when rats were treated in adulthood with the highest accumulative dose tested (12 pulses of 5 mg/kg over 4 days: 60 mg/kg) of psychostimulant drugs, Pearson’s correlation coefficients were calculated to test for possible association between variables. The level of significance was p  0.05.

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3. Results 3.1. Comparative effects of amphetamine-like psychostimulants on body weight at different developmental ages As shown in Fig. 2a, rat’s weight gain did not change throughout the duration of BrdU administration (PND 27–29) and during the experimental treatment with MDMA (see also García-Cabrerizo and García-Fuster, 2015), methamphetamine or D-amphetamine (1 or 4 days) when compared to control treated rats during adolescence (PND 33–36) as evaluated using two-way repeated measures ANOVAs. However, when these drugs were administered at a later developmental age such as young adulthood (PND 48–50 and PND 54–57), the administration of methamphetamine or Damphetamine, but not MDMA (see also García-Cabrerizo and García-Fuster, 2015), reduced rat’s weight gain (Fig. 2b) as demonstrated by a Treatment x Day of Treatment interaction (METH: F12,90 = 4.97, p < 0.001; D-AMPH: F12,90 = 11.74, p < 0.001). Post-hoc analysis revealed reduced weight gain during the repeated exposure to 4 days of methamphetamine (PND 57, p < 0.05) or D-amphetamine (from PND 55–57, p < 0.05) when compared to control treated rats. These results support the previously described anorexigenic role of methamphetamine and D-amphetamine (e.g., Carvalho et al., 2012; Steinkellner

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et al., 2011), while extending the findings to show that these effects occurred only in young adult, as compared to adolescent rats, and were dependent on the accumulative dose (i.e., number of pulses) administered. 3.2. Basal hippocampal cell genesis at different developmental ages Basal hippocampal cell genesis differences were observed between PND 37 and PND 58 when combining and comparing all control groups (C-MDMA, C-METH, C-D-AMPH; Fig. 3). The overall quantification of Ki-67+ and BrdU+ cells by area (mm2) revealed higher rates of recent cell proliferation (+39%, Fig. 3a) and higher survival of 8–10 days old cells (+45%, Fig. 3b) at the earlier time point in adolescence (PND 37) as compared to young adult (PND 58) rats. These age-related differences in cell genesis were observed for all control groups separately, even though there were significant differences in the number of both Ki-67+ and BrdU+ cells among them (i.e., see individual boxes within Fig. 3). Given these basal differences in cell genesis among supposedly identical treated control groups, from now on and for comparative purposes, the effect of each psychostimulant drug will be compared to its respective PND control group (% change).

Fig. 2. Comparative effects of 1 and 4 days of amphetamine-like psychostimulants (MDMA, methamphetamine: METH, D-amphetamine: D-AMPH) (accumulative dose of 15 vs. 60 mg/kg; n = 6 per age and treatment group) on rat body weight during (a) adolescence and (b) young adulthood. Notably, MDMA results have been adapted from published data (García-Cabrerizo and García-Fuster, 2015). PND: post-natal day.

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Fig. 3. Hippocampal cell genesis at different developmental ages. The highest rates of (a) cell proliferation and (b) cell survival (8–10 days old cells) were observed during adolescence as shown by the quantitative analysis of Ki–67 + cells or BrdU + cells per area (mm2) in the left dentate gyrus. Columns are means  SEM of the total number of quantified cells by area in the dentate gyrus for each control group at each age of study (PND 37 and PND 58, n = 6 per age and control group). *** p < 0.001 when compared to PND 58 (Student’s t-test). Representative immunohistochemistry images of (c) Ki-67+ and (d) BrdU+ cells at PND 37 and PND 58. The bigger images were taken in a light microscope using a 40 objective lens to illustrate the anatomy of the dentate gyrus. To observe particular labeled cells, the left-bottom corner of each example show a representative image taken using a 63 objective lens. Scale bar = 30 mm. C-MDMA: control-MDMA group; C-METH: control-methamphetamine group; C-D-AMPH: controlD-amphetamine group.

3.3. Comparative effects of amphetamine-like psychostimulants on hippocampal cell genesis at different developmental ages When evaluating recent cell proliferation, the results showed that an accumulative dose of 15 mg/kg (3 pulses of 5 mg/kg over 1 day) of MDMA, methamphetamine or D-amphetamine was not sufficient to induce changes in the number of Ki-67+ cells/area in the hippocampus of adolescent (PND 37) or young adult (PND 58) rats (Fig. 4a). However, an accumulative dose of 60 mg/kg (12 pulses of 5 mg/kg) over the course of 4 days of either MDMA or methamphetamine, but not D-amphetamine, reduced the number of Ki-67+ cells/area in the hippocampus of adolescent (PND 37: MDMA, 25  6%, p < 0.05) and young adult (PND 58: MDMA, 21  4%, p < 0.05; METH, 26  4%, p < 0.01) rats (Fig. 4b). When evaluating the survival of cells that were labeled with BrdU prior to any psychostimulant drug treatment (i.e., 8–10 days old cells at the time of analysis), an accumulative dose of 15 or 60 mg/kg of MDMA, methamphetamine or D-amphetamine did not induce changes in the hippocampus of adolescent rats (PND 37; Fig. 4c and d). However, the number of BrdU+ surviving cells was altered when these psychostimulant drugs were administered during young adulthood (PND 58; see Fig. 4c and d). In particular, MDMA reduced cell survival following 60 mg/kg (MDMA, 23  5%, p < 0.05) of drug administration (Fig. 4c and d). 3.4. Comparative effects of amphetamine-like psychostimulants on hippocampal BDNF content at different developmental ages Since BDNF is initially synthesized as a precursor protein (proBDNF), and then proteolytically processed into mature BDNF (mBDNF) (Lu, 2003), both protein forms were measured in this study. An accumulative dose of 15 mg/kg of MDMA, methamphetamine or D-amphetamine was not sufficient to induce changes in the content of proBDNF and mBDNF in the hippocampus of adolescent (PND 37) or young adult (PND 58) rats (Fig. 5a–c). However, an accumulative dose of 60 mg/kg over the course of 4 days of MDMA, methamphetamine or D-amphetamine, altered

hippocampal BDNF (Fig. 5d–f). In particular, MDMA reduced hippocampal proBDNF (PND 37: 20  2%, p < 0.05; PND 58: 21  5%, p < 0.05) and mBDNF (PND 37: 24  3%, p < 0.05; PND 58: 23  4%, p = 0.08). On the contrary, methamphetamine and Damphetamine increased hippocampal proBDNF (METH: +22  4%, p < 0.05; D-AMPH: +35  12%, p < 0.05) and mBDNF (METH: +27  12%, p = 0.07; D-AMPH: +43  10%, p < 0.05) but only at PND 58 (Fig. 5d–f). 3.5. Hippocampal cell survival correlates with mBDNF content in young adult rats treated 4 days with amphetamine-like psychostimulants: possible role of 5-HT2C receptors As the main effects for all amphetamine-like drugs on cell genesis markers were observed at PND 58 following treatment with the highest accumulative dose (12 pulses of 5 mg/kg over the course of 4 days: 60 mg/kg), Pearson’s correlation coefficients were calculated for all drug-treated rats to test for possible association between variables (i.e., Ki-67, BrdU, proBDNF, mBDNF, 5-HT2C). Only two pairs of associations came out statistically significant. The number of surviving cells (i.e., BrdU+ cells/area) paralleled the contents of mBDNF in the hippocampus of young adult rats (PND 58) that received the highest dose (60 mg/kg) of amphetamine drugs (r = 0.492, n = 15, p < 0.05; Fig. 6a). Interestingly, in the same rats the contents of mBDNF positively correlated with that of 5HT2C-receptor (r = 0.713, n = 16, p < 0.01; Fig. 6b). In fact, an accumulative dose of 60 mg/kg in young adult rats (from PND 54–57) altered 5-HT2C-receptor in the hippocampus for MDMA (–60  7%, n = 5, p < 0.05; see García-Cabrerizo and García-Fuster, 2015), and methamphetamine (+34  10%, n = 6, p < 0.05, one tail ttest), but not for D-amphetamine (+25  12%, n = 6, p > 0.05). 4. Discussion The main results of this study suggest that the effects of amphetamine-like drugs on cell genesis depend on the rat’s developmental age, with the young adult period being more

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Fig. 4. Comparative effects of amphetamine-like psychostimulants on hippocampal cell genesis at different developmental ages (PND 37 vs. PND 58, n = 6 per age and treatment group, except the group treated with MDMA 60 mg/kg, n = 5). (a–b) Cell proliferation as measured by the number of Ki–67 + cells by area (mm2) in the dentate gyrus affected by an accumulative drug dose of (a) 15 mg/kg (3 pulses of 5 mg/kg) or (b) 60 mg/kg (12 pulses of 5 mg/kg) at PND 37 and PND 58. (c–d) Cell survival as measured by the number of BrdU + cells by area (mm2) in the dentate gyrus affected by an accumulative drug dose of (c) 15 mg/kg or (d) 60 mg/kg at PND 37 and PND 58. * p < 0.05 and ** p < 0.01 when compared with the corresponding age-control group (Student’s t-test). Notably, some control-drug pairwise comparisons might not be statistically significant as the SEM value for each PND control group has to be considered and is not represented in the graphs.

sensitive than the early adolescent one. MDMA and methamphetamine, but not D-amphetamine impaired hippocampal cell genesis in young adult rats. These effects were also dependent on the accumulative dose tested, as they were only observed following 12 pulses of 5 mg/kg (60 mg/kg over the course of 4 days). On the contrary, D-amphetamine increased both pro and mature BDNF forms in the hippocampus of young adult rats suggesting certain degree of neuroprotection and/or survival signals. Interestingly, mBDNF protein regulation paralleled hippocampal cell survival and 5-HT2C-receptor content in young adult rats treated with the highest dose tested of psychostimulant drugs. The enhanced rates of cell proliferation and cell survival observed in adolescent rats in comparison to young adult rats is likely due to the developmental process. Similarly, other studies have shown higher hippocampal neurogenesis rates in adolescence as compared to adulthood (e.g., He and Crews, 2007; GarcíaCabrerizo et al., 2015), reflective of the great level of neuroplasticity during adolescence (i.e., overproduction of axon and synapses; Spear, 2004) that might contribute to brain maturation and the transition to adulthood. These age-related basal differences in cell genesis were observed for all control groups separately (i.e., see individual boxes for each control group, Fig. 3). However, there were significant basal differences among supposedly identical control-treated groups. This might be explained by the fact that each experimental drug treatment (i.e., MDMA, methamphetamine, D-amphetamine) was done at different points in time, with different colonies provided by the

vendor (e.g., Fitzpatrick et al., 2013) and/or particular environmental conditions (Schmidt and Duman, 2007), and points out the relevance of controlling variables when studying hippocampal cell genesis. Interestingly, the observed decline in cell genesis from early adolescence to young adulthood represents a critical window of either vulnerability (i.e., leading to more damage in adulthood) or opportunity (i.e., better adaptation) to study hippocampal remodeling (Dahl, 2004) in response to challenges, such as drug exposure. In particular, MDMA has been shown to regulate different stages of hippocampal neurogenesis depending on the mode, dose and age of drug administration as well as the species studied. For instance, when MDMA was administered during adolescence (i.e., every other day from PND 31–39), lower doses (total accumulative dose of 5 or 10 mg/kg) did not alter progenitor cell proliferation or survival as compared to a higher total accumulative dose (20 mg/ kg) that increased cell proliferation yet diminished cell survival during withdrawal (Catlow et al., 2010). Moreover, 20 mg/kg of MDMA exposure during adolescence caused neurotoxic alterations in the hippocampus by reducing survival of neuronal precursors (Hernández-Rabaza et al., 2010). When MDMA was administered in adult rats a total accumulative dose of 40 mg/kg showed no effect on cell proliferation but decreased survival during withdrawal (Hernández-Rabaza et al., 2006), while in mice MDMA decreased cell proliferation in a dose dependent manner (doserange: 1.25–40 mg/kg, daily injections for up to 30 days; Cho et al., 2007). In this context, an accumulative dose of 60 mg/kg of MDMA

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Fig. 5. Comparative effects of amphetamine-like psychostimulants on hippocampal BDNF protein content at different developmental ages (PND 37 vs. PND 58) (n = 6 per age and treatment group, except the group treated with MDMA 60 mg/kg, n = 5). (a–c) Effects of an accumulative dose of 15 mg/kg (3 pulses of 5 mg/kg) on (a, c) proBDNF and (b–c) mBDNF protein content at PND 37 and PND 58. (d–e) Effects of an accumulative dose of 60 mg/kg (12 pulses of 5 mg/kg) on (d, f) proBDNF and (e–f) mBDNF protein content at PND 37 and PND 58. * p < 0.05 when compared with the corresponding age-control group (Student’s t-test). Notably, some control-drug pairwise comparisons might not be statistically significant as the SEM value for each PND control group has to be considered and is not represented in the graphs. (c, f) Representative immunoblots depicting labeling of proBDNF (32 kDa) and mBDNF (14 kDa) are shown for each set of experiments.

(12 injections of 5 mg/kg over the course of 4 days) was required to impair cell genesis in the hippocampus of both adolescent (i.e., cell proliferation) and young adult (i.e., cell proliferation and survival) rats, suggesting in line with the existing literature, a neurotoxic effect of MDMA in the hippocampus. Similarly, methamphetamine reduced cell proliferation in the hippocampus of young adult rats following an accumulative dose of 60 mg/kg (12 injections of 5 mg/ kg over the course of 4 days) while induced a non-significant decrease in cell survival. Moreover, no significant effects on cell genesis were observed when methamphetamine was administered earlier during adolescence or at a lower accumulative dose (i.e., 15 mg/kg administered in 3 pulses of 5 mg/kg). Comparably, prior reports in adult rats suggested that the amount of methamphetamine consumed following a self-administration paradigm was relative to the magnitude of reduction in hippocampal neurogenesis (Mandyam et al., 2008; Yuan et al., 2011). Compared to MDMA and methamphetamine previously discussed data, only a few studies have explored the effect of Damphetamine on adult hippocampal neurogenesis. On one hand, Barr et al. (2010) showed no effect on cell proliferation and survival following repeated D-amphetamine treatment (2.5 mg/kg, i.p., daily for two weeks) in adult rats. However, another study that

explored the effect of chronic D-amphetamine administration (0.25, 0.5 or 2 mg/kg, i.p., twice daily) from early adolescence (PND 28) to adulthood (PND 71) showed no effect on hippocampal cell proliferation, but increased cell survival and neuronal differentiation in mouse brain (Dabe et al., 2013). Our results showed that Damphetamine did not alter cell proliferation or survival in the hippocampus of rats at any age studied, although D-amphetamine increased by 17–48%, without reaching statistical significance, the number of BrdU cells in young adult rats. These overall comparative studies demonstrated that experimenter-delivered MDMA or methamphetamine, but not D-amphetamine, impaired hippocampal cell genesis depending on the accumulative dose administered (i.e., number of 5 mg/kg pulses), in young adult rats. Thus, these results extend previous reports on adolescent insensitivity (i.e., better adaptation) to amphetamine-psychostimulant drugs and suggest for adult rats certain degree of hippocampal damage that may mediate some of the addiction behaviors dependent on this brain region. For example, much research has been done on the relationship between the observed negative effects of stimulant drugs on hippocampal neurogenesis and their influence on memory dysfunction (reviewed in Canales, 2010). In fact, levels of neural progenitors in the hippocampus have

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Fig. 6. Hippocampal cell survival correlates with mBDNF content in young adult rats treated with 12 pulses of 5 mg/kg of amphetamine-like psychostimulants over the course of 4 days: Possible role of 5-HT2C receptors. (a) Scatter plot depicting a significant positive correlation between the number of BrdU + cells and mBDNF protein content in the hippocampus after 4 days of amphetamine-like drug treatment and expressed as percentage of the corresponding PND-control group. Each circle represents a different treated rat. The solid line is the best fit for the correlation (r = 0.492, n = 15, p < 0.05). The dotted curves indicate the 95% confidence interval for the regression line. (b) Scatter plot depicting a significant positive correlation between mBDNF and 5-HT2C receptor protein content in the hippocampus after 4 days of amphetamine-like drug treatment and expressed as percentage of the corresponding PND-control group. Each circle represents a different treated rat. The solid line is the best fit for the correlation (r = 0.713, n = 16, p < 0.01). The dotted curves indicate the 95% confidence interval for the regression line.

been shown to predict memory impairment and relapse to drug seeking (Recinto et al., 2012). Contrarily, the inexistent harmful effects of D-amphetamine on hippocampal cell genesis in the present results goes along with the existing literature and its use as a cognitive enhancer (Greely et al., 2008). The functional significance and the mechanisms by which these amphetamine-drugs regulate cell genesis remains elusive. As stated earlier in the introduction, BDNF is critical for the function and survival of neurons (Mattson et al., 2004). Prior experiments suggested a time-dependent and region-specific modulation of BDNF by different dose regimens of these amphetamine-like psychostimulant drugs (see below). Similarly, the present results suggest that these drugs regulate BDNF forms depending on the dose administered (i.e., effects seen following 12 pulses of 5 mg/kg: 60 mg/kg total) and the age of development (i.e., mainly in young adult rats). In particular, while MDMA reduced hippocampal BDNF protein forms, methamphetamine and D-amphetamine increased them. Comparably to the present results, the hippocampus showed temporal decreases or unaltered BDNF content in adolescent (Ádori et al., 2010) or adult (Martínez-Turillas et al., 2006; Hemmerle et al., 2012) rat brain by MDMA. Additionally, methamphetamine effects on increasing BNDF have been shown at different developmental ages. For example, methamphetamine increased BDNF mRNA in the hippocampus during development (Grace et al., 2008) and in a time-dependent manner in adult rats (Braun et al., 2011). Moreover, at the protein level, extended access methamphetamine enhanced expression of mBDNF in the dorsal

and ventral hippocampus (Galinato et al., 2015). However, when revisiting the published studies evaluating D-amphetamine effects on BDNF the results seemed to be inconsistent. Some studies showed decreased BDNF mRNA expression in the hippocampus following D-amphetamine administration in adolescent but not adult rats (Banerjee et al., 2009; 0.5 mg/kg dose) or decreased protein in adult rats (Fuller et al., 2015; 12 escalating doses given in 4 days for a total 75 mg/kg accumulative dose). Yet, similarly to the present results, repeated treatments in adult rats with Damphetamine (5 mg/kg/day for 5 days) increased BDNF expression and production in multiple brain regions (Meredith et al., 2002), including increased levels of hippocampal BDNF (Griesbach et al., 2008; 1 mg/kg/day for 7 days). Therefore, while MDMA decreased BDNF content in the hippocampus, D-amphetamine increased it. In this context, diminished BDNF levels could be associated with stress (i.e., MDMA drug effects on serotonergic nerve endings; for possible link between BDNF, serotonin and neurogenesis see review Foltran and Diaz, 2016) while increased levels could suggest neural adaptations following brain toxicity (i.e., neuroprotection following methamphetamine drug effects; Dluzer, 2004) or survival-signals (i.e., D-amphetamine drug effects). An association between cell survival and mBNDF was observed by a correlation analysis in the hippocampus of young adult rats treated with 12 pulses of 5 mg/kg (i.e., the highest dose tested) of these psychostimulant drugs, suggesting mBDNF might engage to assist with changes in hippocampal plasticity. Similarly, a recent study showed that the survival of adult brain progenitors was

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associated with altered BDNF signaling (Somkuwar et al., 2015). Interestingly, besides BDNF, serotonin has been shown to participate in regulating cell genesis and neuronal survival in the adult brain (Mattson et al., 2004; Foltran and Diaz, 2016). In fact, the serotonin system is known to undergo substantial development during adolescence (Chen et al., 1997) with 5-HT2C receptors present at higher densities in adult hippocampus (reviewed in Berumen et al., 2012). Moreover, a recent study showed MDMA administration decreased hippocampal content of 5-HT2C receptors in young adult rats (García-Cabrerizo and GarcíaFuster, 2015). In this context, the present results suggest an association and a possible role for 5-HT2C-receptors in mBDNFsurvival regulation as observed by the positive correlation between them. Interestingly, 5-HT2C-receptor activity has been shown to promote proliferation and differentiation of neuronal determined progenitors cells (Klempin et al., 2010). 5. Conclusions The present results demonstrate certain degree of hippocampal damage exerted by MDMA or methamphetamine in the hippocampus of young adult rats, that may mediate some of the addiction-like behaviors dependent on this brain region and extend previous reports on adolescent insensitivity (i.e., better adaptation) to amphetamine-like drugs. Moreover, D-amphetamine increased both pro and mature BDNF forms in the hippocampus of young adult rats proposing certain degree of neuroprotection and/or survival signals. Interestingly, mBDNF regulation paralleled hippocampal cell survival and 5-HT2Creceptor content when young adult rats were treated with amphetamine-like drugs. Future studies will aim at evaluating the long-term consequences to drug-induced brain changes of treating rats at this period of vulnerability. Role of funding source This study was supported by Fundación Alicia Koplowitz and by Delegación del Gobierno para el Plan Nacional sobre Drogas (grant number 2012/011, Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain) to MJG-F. The study was also funded by RETICSRTA (RD12/0028/0011; Instituto de Salud Carlos III, MINECO/ FEDER, Spain). RG-C was supported by a pre-doctoral fellowship (Consejería de Innovación, Investigación y Turismo del Gobierno de las Islas Baleares y del Fondo Social Europeo). MJG-F is a ‘Ramón y Cajal’ Researcher (MINECO-UIB). Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors would like to thank Drs. Huda Akil and Stanley J. Watson (University of Michigan, Ann Arbor, MI, USA), who kindly provided Ki-67 and BrdU antibodies. The authors also thank Mr. Antonio Crespo for technical assistance. References Ádori, C., Andó, R.D., Ferrington, L., Szekeres, M., Vas, S., Kelly, P.A., Hunyady, L., Bagdy, G., 2010. Elevated BDNF protein in cortex but not in hippocampus of MDMA-treated Dark Agouti rats: a potential link to the long-term recovery of serotonergic axons. Neurosci. Lett. 478, 56–60. Banerjee, P.S., Aston, J., Khundakar, A.A., Zetterström, T.S., 2009. Differential regulation of psychostimulant-induced gene expression of brain-derived neurotrophic factor and the immediate-early gene Arc in the juvenile and adult brain. Eur. J. Neurosci. 29, 465–476.

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